WO2003059062A1

WO2003059062A1 - Virucidal disinfectant

Info

Publication number: WO2003059062A1
Application number: PCT/EP2003/000378
Authority: WO
Inventors: Florian Lichtenberg; Michael LÜTZELER; Volker Ranft
Original assignee: Lonza Ag
Priority date: 2002-01-18
Filing date: 2003-01-16
Publication date: 2003-07-24
Also published as: EP1469729A1; EP1469729B1; AU2003202570A1; AU2008229805A1; ES2554102T3; JP4647912B2; PL372273A1; US20050089496A1; NZ534534A; JP2005514427A; CA2484634A1; DK1469729T3; CA2484634C; US8221733B2; ZA200405828B

Abstract

Disclosed is the use of disinfectant compositions as a virucide, particularly against polio viruses, containing a) at least one amine and/or quaternary ammonium salt of general formula (Ia) or (Ib), in which R1 represents C6-18 alkyl, R2 represents benzyl or C6-18 alkyl, R3 represents C1-18 alkyl or -[(CH2)2-O]nR6, with n = 1-20, R4 and R5 independently represent C1-4 alkyl, R6 represents hydrogen or optionally substituted phenyl, and A- represents a monovalent anion or an equivalent of a polyvalent anion of an inorganic or organic acid; and b) at least one alkanolamine of general formula (II), in which n and optionally m and o independently have the value 2 or 3 and x and y independently have the value 0 or 1, or a corresponding salt; at a mass ratio I:II of 20:1 to 1:20. Said compositions also have good bactericidal and fungicidal qualities, even when used at low concentrations.

Description

Virucidal disinfectant

The invention relates to the use of synergistic disinfectant compositions based on A inen and / or quaternary ammonium salts as virucidal agents, in particular against polioviruses.

Numerous disinfecting and preservative compositions based on amines and / or quaternary ammonium salts are known. However, these have in general, especially at higher dilutions, an unsatisfactory activity against fungi such. B. Aspergillus niger and viruses (especially against highly resistant viruses such as polio virus).

The object of the present invention was therefore to provide disinfectant compositions based on amines and / or quaternary ammonium salts which, even at high dilution, have good activity against fungi and in particular against viruses.

According to the invention this object is achieved by the use according to claim 1.

The earlier application PCT / EP 01/10754 (published as WO 02/23990 Al) describes disinfectant compositions based on amines and / or quaternary ammonium salts and alkanolamines and their fungicidal properties. It has now surprisingly been found that such disinfectant compositions also have pronounced virucidal properties and in particular also a good activity against highly resistant viruses such as polioviruses. They are also effective against other picomaviruses such as ECHO viruses or corresponding animal pathogenic viruses such as ECBO viruses and against parvoviruses such as Canine Parvovirus.

The compositions include amines and / or quaternary ammonium salts of the general formula

wherein R ¹ CE_ ₁₈ - alkyl, R ² is benzyl or C _<5 _ ₁₈ - alkyl R ³ is C 8 alkyl or - [(CH ₂₎ ₂ -O] "R ⁶ with n = 1-20 R ⁴ and R ⁵ independently Cι- ₄ alkyl R ⁶ is hydrogen or optionally substituted phenyl and a ^~ is a monovalent anion or one equivalent of a polyvalent anion of an inorganic or organic acid; and at least one alkanolamine of the general formula

KCH ₂ ) _m O ^ H

HO- (CH ₂ ) -N (D),

| (CH ₂ ) -O ^ H

wherein n and, if present, m and o are independently 2 or 3 and x and y are independently 0 or 1, or a corresponding salt; in the mass ratio 1:11 from 20: 1 to 1:20.

By alkyl are here and hereinafter understood in each case to be linear or branched alkyl groups of the stated carbon numbers, but preferably linear alkyl groups and particularly preferably those having an even number of carbon atoms. In particular, this also includes the homolog mixtures derived from natural raw materials, such as "cocoalkyl" for example.

By substituted phenyl in particular with one or more d- to understand ₈ alkyl groups and / or chlorine atoms substituted phenyl groups.

As anion A ^~ are basically all inorganic or organic anions, in particular halide such as chloride or bromide or anions lower Carboxylic acids such as acetate, propionate or lactate.

The amine or quaternary ammonium salt (Ia / Ib) is preferably N, N-bis (3-aminopropyl) -dodecylamine, N, N-bis (3-aminopropyl) octylamine, a didecyldimethylammonium salt, dioctyldimethylammonium salt, octyl-decyl dimethylammonium salt, dicoco-alkyl-dimethylammonium salt, cocoalkyl-dimethyl-poly (oxyethyl) ammomium salt, dicocoalkylmethyl-poly (oxyethyl) ammonium salt, decyl-dimethyl-poly (oxyethyl) -momomium salt, didecyl-methyl-poly (oxyethyl) ammonium salt, octyl -dimethyl-poly (oxyethyl) ammonium salt, dioctylmethyl-poly (oxyethyl) ammonium salt, cocoalkyldimethylbenzylammonium salt, benzyldodecyldimethylammonium salt or benzyldimethyl-poly (oxyethyl) ammonium salt or a mixture of two or more of these compounds. Particularly good results were achieved with didecyl dimethyl ammonium salts.

Suitable alkanolamines (II) are in principle all ethanol and propanolamines, in particular monoethanolamine, diethanolamine, triethanolamine and 3-amino-1-propanol. It is of course also within the scope of the invention to use mixtures of the compounds mentioned. Particularly good results were obtained with the compounds having a primary amino group, namely with monoethanolamine and 3-amino-1-propanol.

The mass ratio of amine (Ia) or quaternary ammonium salt (Ib) to alkanolamine (II) is preferably in the range from 1: 5 to 5: 1.

The disinfectant compositions used according to the invention preferably contain water as solvent, optionally in combination with an organic solvent.

The disinfectant compositions used according to the invention preferably also contain one or more auxiliary substances from the group consisting of organic solvents, surfactants, complexing agents, fragrances and dyes.

A preferred use of the disinfectant compositions is surface and instrument disinfection. Further preferred fields of use are laundry disinfection and hand disinfection.

Another preferred use of the disinfectant compositions is the use in chemical toilets such as on board aircraft and vehicles.

The following examples illustrate the embodiment of the invention, without it being a limitation to the described embodiments. All quantities are in mass%, unless stated otherwise. The test germ used in each case was Aspergillus niger ATCC 16404. Unless stated otherwise, the efficacy was determined according to the procedure specified in CEN 1275.

example 1

A disinfectant cleaner formulation (concentrate) was prepared from:

5.0% didecyldimethylammonium chloride (50% solution) 2.0% N, N-bis (3-aminopropyl) dodecylamine

5.0% monoethanolamine

5.0% Genapol ^® T250 (Talgfettalkoholpolyglycolether, 25 mol of ethylene oxide)

0.5% sodium metasilicate

0.5% 2.0% Νatriumcarbonat methylglycinediacetic acid trisodium salt (Trilon ^® M; 40% solution) Water ad 100%

The effectiveness was determined with a dilution (1 part concentrate, 99 parts water) at 20 ° C and 15 min contact time. The decadic logarithm of germ count reduction was 4.1. Comparative Example 1

The procedure was as in Example 1, but with the difference that the monoethanol was replaced by the same amount of water. Under the same test conditions, the formulation was virtually ineffective.

Example 2

A disinfectant formulation (concentrate) was prepared from:

4.9% N, N-bis (3-aminopropyl) dodecylamine

4.0% monoethanolamine

2.0% Genapol ^® T250 (Talgfettalkoholpolyglycolether, 25 mol of ethylene oxide) 5.0% Hostapur ^® SAS 30 (C ₁₃ _ι ₇ secondary o-alkanesulfonic acid, Νatriumsalz)

2.0% ethylenediaminetetraacetic acid tetrasodium salt (40% solution)

0.7%) ethylenediaminetetraacetic acid water ad 100%

Efficacy was determined by dilution (1 part concentrate, 199 parts water) at 20 ° C and 15 minutes contact time. The decadal logarithm of germ count reduction was 4.3.

Example 3

A desmfektionsmittelformulierung (concentrate) was prepared from:

4.2%) N, N-bis (3-aminopropyl) dodecylamine 2.0% didecylmethyl poly (oxyethyl) ammonium propionate (BARDAP 26)

4.0%) monoethanolamine

2.0% Genapol ^® T250 (Talgfettalkoholpolyglycolether, 25 mol of ethylene oxide)

5.0% Hostapur ^® SAS 30 (C ₁₃ _ ₁ secondary n-alkane sulfonic acid, Νatriumsalz) 2.0% ethylenediaminetetraacetic acid tetrasodium salt (40% solution) 0.7% ethylenediaminetetraacetic acid 4.0% butyldiglycol

Water ad 100%

The efficacy was determined with a dilution (1 part concentrate, 199 parts water) at 20 ° C and 15 min contact time. The decadal logarithm of germ count reduction was> 4.4. In addition, the efficacy was still determined according to the procedure specified in CEN 1650 with a contact time of 15 minutes, a concentration of 1.0%, a water hardness of 30 ° fH and an organic load of 0.3% albumin. The decadal logarithm of germ count reduction was> 4.4.

Examples 4-19

Aqueous solutions of 0.5% alkanolamine (II) and 0.25% amine or quaternary ammonium salt (Ia / Ib) were prepared and tested according to the procedure specified in CEN 1275. The results are summarized in the following Table 1.

Table 1

For comparison, all compounds listed in Table 1 were tested as individual substances in 0.5% solution. None of these compounds showed a pronounced fungicidal action (log ₁₀ germ reduction <2). Example 20

A disinfectant formulation (concentrate) was prepared from:

9.9% didecyl dimethylammomum chloride (70% solution)

8.0% monoethanolamine

5.0% potassium carbonate (anhydrous)

6.0% ethylenediaminetetraacetic acid tetrasodium salt (Trilon B ^®; 40% solution) Water ad 100%

Example 21

The concentrate described in Example 20 was in 6% dilution m

Suspension test with a contact time of 30, 60 and 120 minutes tested for activity against poliovirus type 1 (Mahoney strain).

Test method: The test was carried out in accordance with the "Guideline of the Federal Health Office and the German Association for the Control of Virus Diseases for the Testing of Chemical Disinfectants for Activity against Viruses" (Bundesgesundheitsbl., 1982, 25, 397) "Dulbecco's Modified Eagle's Medium" to which 10% fetal calf serum and 10 U / ml penicillin and 10 μg / ml streptomycin had been added. After inoculation of the tissue culture with polio virus, the tissue culture medium contained only 3% fetal calf serum. After almost complete detachment of polio-infected cells, the virus suspension was purified by centrifuging off cells and cell components (3000 g, 15 min). Since the medium of the cell culture contained 3% fetal calf serum, a small protein load was also present in the disinfectant test in the test batches with double-distilled water.

For the disinfectant test, 1 part of the virus suspension was mixed with 8 parts of a 7.5% dilution of the disinfectant concentrate (corresponding to a final concentration of 6%) and 1 part of double-distilled water. or 2% serum albumin or fetal calf serum and incubated for 30, 60 and 120 minutes at 20 ° C. Then, the effect of the disinfectant was stopped by 100-fold dilution with cold medium containing no fetal calf serum. In each case 1 ml of this dilution (corresponding to a dilution of the virus suspension to 10 ^-3 ) and further decadal dilution stages were each 2 small wells of 6-well multiwell plates (Becton Dickinson Labware, Lincoln Park, NJ, type Falcon ™ 353046), forming a dense cell lawn of Vero cells, inoculated. After 1 h adsorption time at room temperature, the supernatant liquid was aspirated. Then, the cell dish of the dishes was mixed with 2 ml of 2% agarose liquefied by cooking (Serva high EEO, cat # 11397) mixed with double concentrated medium with 5% fetal calf serum in a ratio of 1: 1 and in a water bath was cooled to 40 ° C, overlaid. After solidification of the agarose at room temperature, the plates were incubated at 37 ° C in the CO ₂ incubator for 2 days. The infectivity of the virus suspension was tested in the plaque test. Each area of destroyed cells corresponds to an infectious unit of the poliovirus. The number of plaques thus indicates the number of infectious virus particles present in a given dilution of the experimental batch. The plaques are visualized by staining with 1.0 ml of a solution of 0.1% Brilliant Blue R (Sigma, Cat # B0149) in an aqueous solution containing 20% methanol and 5% acetic acid for 30 min. The unstained plaques are then markedly different from the blue stained cell lawn. From each of two batches of a dilution, an average of the plaque count was calculated. The "viral controls" used to determine the starting concentration of the virus were batches in which the disinfectant was replaced by the same volume of bidistilled water, and the virus concentration was used as a reference for calculating the virus-inactivating effect of the tested disinfectants "Toxicity controls" to detect any damage to the tissue culture cells by the disinfectant were used in approaches in which the virus suspension was replaced by the same volume of bidistilled water. These batches were diluted 1: 100 and 1: 1000 (corresponding to a dilution of the virus suspension of 10 ^-3 and 10 ⁴ in the disinfectant assay, respectively) with medium without fetal calf serum and then, like the approaches for testing the disinfecting effect for 1 h the Tissue culture was added and then stripped off. After 2 days of incubation at 37 ° C, it was checked by staining whether the cell lawn had been damaged by the disinfectant. As a reference to the resistance of the test virus and to comparability with other studies, a "formaldehyde control" was carried out by mixing 1 part of the virus suspension with 4 parts of phosphate buffered saline (0.1 M, pH 7, "Dulbecco's PBS") the same amount of formalin solution containing 1.4 g of formaldehyde in 100 ml solution (final concentration: 0.7 g HCHO / 100 ml). After 5, 15 and 60 min contact time, the effectiveness of the formaldehyde was stopped by dilution to 1: 100, as in the disinfectant test, and the residual infectivity of the poliovirus was determined in further decadal dilutions in the plaque test.

Results: Control experiments: The "virus control" in the batch with bidistilled water gave a virus concentration of 1.6-10 infectious units / ml, in the beginning with serum albumin 1.2-10 infectious units ml and in the batch with fetal calf serum 1.0- 10 ⁸ infectious units / ml. The "toxicity control" showed a slight damage to the cell lawn after dilution of the test batch to 1: 100 (corresponding to a dilution of the virus suspension of 10 ^-3 ). At a dilution of 1: 1000, no toxicity was evident. Thus, under the test conditions, a decrease in viral concentration under disinfectant action can be followed up to a virus concentration of 5-10 ³ infectious units / ml in the virus suspension (no plaque is visible in both 10 ^"4 dilution dishes) and at a starting concentration of At least 10 infectious virus particles / ml, a decrease in the virus concentration over at least 4.5 orders of magnitude is observable.Since the test guideline to demonstrate the effectiveness of a disinfectant only requires a decrease in the virus concentration by at least 4 orders of magnitude, the fulfillment of this condition can be checked with the selected experimental approach. In the batch with 0.7% formaldehyde, after 5 min exposure time, a virus concentration of 1.05-10 ⁶ / ml, after 15 min 1 -10 ³ / ml and after 60 min <5-10 ² / ml were measured Expected values confirming the results of previous experiments: 0.7% formalin is usually able to reduce the concentration of poliovirus by more than 4 orders of magnitude within 30 minutes.

Effectiveness of the disinfectants against poliovirus: After 30, 60 and 120 min exposure to 6% dilution of the disinfectant composition from Example 20, plaque was no longer observed in the preparation with fetal calf serum at the virus dilution 10 ^{"4 in} each test dish the disinfectant treatment had a virus concentration of <5-10 infectious units / ml, and this result was found in both low (bi-distilled and mixed medium) and medium (2% serum albumin) and high (fetal calf serum) protein load. Thus, in comparison with the control determination without disinfectant, a decrease in the virus concentration by at least 4.5 × 10 _2- steps or powers of 10 was achieved, thus fulfilling the precondition for the registration as an instrument disinfectant in the Federal Republic of Germany.

Example 22

Efficacy against ECBO viruses:

The concentrate described in Example 20 was based on the guidelines of the (German) Federal Health Office and the German Association for the Control of Viral Diseases eV for testing chemical disinfectants for effectiveness against viruses (Bundesgesundheitsbl., 1982, 25, 397-398, Comment: Bundesgesundheitsbl. 1983, 26, 413-414) in the quantitative suspension test for its virucidal properties against the ECBO virus strain LCR-4. Dilutions of 1.0%, 3.0% and 5.0% of the concentrate were tested in bidistilled water at exposure times of 15, 30, 60 and 120 minutes. The test temperature was 20 ± 1 ° C, as protein load, fetal calf serum (FCS) or serum albumin (bovine seram albumin, BSA) was used.

To prepare the virus suspension, fetal calf lung cells (FKL 107) were placed in Roux bottles with Minimum Essential Medium (MEM, Eagle) containing approximately 10 PBE (plaque forming units) of the virus (obtained from Dr. W. Herbst, Institute of Hygiene and Health Infectious diseases of the animals at the Justus Liebig University in Giessen) per cell infected and subjected to a threefold freezing / thawing process after the appearance of the cytopathic effect (about 12 h). This was followed by centrifugation at 770 xg for 10 min, which provided the virus suspension as a supernatant. To prepare the inactivation batches, 8 parts by volume of the disinfectant were mixed with virus suspension and double-distilled water (1 part by volume each) in a concentration of l, 25 times the desired concentration. In the tests with protein load, instead of the double-distilled water, one part by volume of FKS (Flow Laboratories) or 2% BSA solution (Behringwerke AG) was used. The inactivation experiments were carried out in closed glass tubes. After the appropriate times, samples were taken to determine residual infectivity.

The infectivity was determined by means of the final dilution titration in the micro procedure. For this purpose, the samples were diluted immediately after their collection with Minimum Essential Medium (MEM), with dilution factors selected as integer powers of 10. Each 100 μl of a diluted sample was transferred to 8 wells of a flat bottom sterile polystyrene plate. Then, in each case, 100 μl of a freshly trypsinized suspension of KOP-R cells (bovine oesopharyngeal tissue, obtained from Dr. R. Riebe, Federal Research Center for Viral Diseases of Animals on the Island of Riems, catalog No. RIE 244) were added. This suspension was adjusted so that approximately 10-15 x 10 ³ cells were in each well. Thereafter, the samples were incubated at 37 ° C in a CO ₂ incubator (5 vol% CO ₂ ). After 5 to 7 days, the infectious dose (IDso / ml) was determined by the method of Spearmann-Kärber. The evaluation of the virucidal activity was carried out by calculating the titer drop compared to the control titrations carried out in parallel (after the longest reaction time). The difference was given as Δlogio ID ₅ o.

To determine the cytotoxicity of the disinfectant, 2 volumes of PBS (phosphate buffered saline) were mixed with 8 volumes of the disinfectant dilution (1.25 fold concentration), diluted appropriately and applied to the cell cultures. The cytotoxic dose was reported as log ₁₀ CDso / ml (in analogy to the ID ₅ o value).

The results of the inactivation experiments are summarized in the following Table 2, those of the cytotoxicity determination in Table 3. Table 2

nd = not performed

Table 3

The results show that the tested composition (concentrate) at an application concentration of 3.0% after 60 min exposure time and at 5.0% after 30 min the efficacy defined in the guideline (Δlog ^ IDso ≥ 4.0; Inactivation of 99.99%) against ECBO viruses. Example 23

Efficacy against Canine Parvovirus:

Dilutions of the concentrate described in Example 20 were tested for efficacy against Canine Parvovirus Type 2 (obtained from Dr. Parrish, Cornell University) in NLFK.

Cells (North Lab Feline Kidney) tested at 22 ° C and 10 min exposure time.

Dilutions 1:35 in desalted or hard (400 ppm AOAC hard water)

Water with 5% organic load (fetal calf serum) showed sufficient virucidal

Effectiveness.

Claims

claims

1. Use of a disinfectant composition comprising a) an amine and / or quaternary ammonium salt of the general formula

(CHANEL R ^J

, /? '+ 4

R-N (Ia) or R-N-R A (Ib),

(CHANH R ^J

wherein R ¹ is C _Ö - _IS - alkyl,

R ² is benzyl or C ₆ -ι ₈ alkyl, R ³ Cι_i ₈ alkyl or - [(CH ₂₎ ₂ -O] "R ⁶ with n = 1-20,

R ⁴ and R ⁵ independently of one another are C 1 - alkyl, R ^{6 is} hydrogen or optionally substituted phenyl and A ^{- is} a monovalent anion or one equivalent of a polyvalent anion of an inorganic or organic acid; and b) at least one alkanolamine of the general formula

wherein n and, if present, m and o are independently 2 or 3 and x and y are independently 0 or 1, or a corresponding salt; in a mass ratio of 1:11 from 20: 1 to 1:20 as a virucidal agent.

2. Use according to claim 1, characterized in that the amine or quaternary ammonium salt is selected from the group consisting of N, N-bis (3-amino-propyl) dodecylamine, N, N-bis (3-aminopropyl) octylamine , Didecyldimethylammonium salts, dioctyldimethylammonium salts, octyldodyldimethylammonium salts, Cocoalkyldimethylbenzylammonium salts and benzyldimethyl-oxethylammonium salts and mixtures of these compounds.

3. Use according to claim 1 or 2, characterized in that the alkanolamine (II) is selected from the group consisting of monoethanolamine, diethanolamine,

Triethanolamine and 3-amino-1-propanol.

4. Use according to one of claims 1 to 3, characterized in that the mass ratio 1:11 is between 1: 5 and 5: 1.

5. Use according to one of claims 1 to 4, characterized in that the disinfectant composition contains water as a solvent.

6. Use according to any one of claims 1 to 5, characterized in that the disinfectant composition additionally one or more excipients from the

Group consisting of organic solvents, surfactants, complexing agents, fragrances and dyes.

7. Use according to one of claims 1 to 6 in the surface and instrument disinfection.

8. Use according to one of claims 1 to 6 in the laundry disinfection.

9. Use according to one of claims 1 to 6 in the hand disinfection.

10. Use according to one of claims 1 to 6 in chemical toilets.

11. Use according to one of claims 1 to 10 against parvo- or picornaviruses, in particular against polioviruses.